Peptide having ability to activate cancer-related gene

ABSTRACT

To provide a cancer diagnostic reagent for determining malignancy of a cancer patient or a cancer cell and a tendency of canceration of a healthy subject, the reagent including a peptide having an ability to activate a cancer-related gene and extracted from cell membrane surfaces of human squamous-cell carcinoma cells or including a synthetic polynucleotide encoding the peptide or a partial amino acid sequence of the peptide.

This application is a division of U.S. patent application Ser. No. 11/486,123 filed on Jul. 14, 2006, now U.S. Pat. No. 7,579,434 issued Aug. 25, 2009, which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to peptides activating a cancer-related gene and derived from cell membrane surfaces of human cancer cells, immunosuppressive agents including the peptides as an effective component, and anticancer agents including antibodies against the peptides as an effective component. The present invention further relates to diagnostic reagents including polynucleotides encoding the peptides for diagnosing malignancy of cancer or tendency of canceration. Furthermore, the present invention relates to methods for manufacturing the peptides.

2. Description of the Related Art

Cancer is a disease caused by cells which started unregulated growth. The cells continue to proliferate and infiltrate into neighboring normal cells to destroy the normal functions. Some of the cells spread by metastasis from their original site to one or more sites elsewhere in the body, leading to a loss of normal cell functions and to depression of functions of organs. Thus, cancer is a disease that leads patients to death. Normal cells proliferate but will stop growing when they come in contact with solid substances (contact inhibition). On the other hand, the contact inhibition property is lost in cancerous cells. Consequently, the cancerous cells continue to grow in their host as long as the host is alive. Among cells extracted from the body, cells having ability for continuing unlimited growth can be established as a cell line. Cancer, in a broad sense, is a state of uncontrolled cell growth due to mutation of a gene, in particular, due to mutation of a gene positively or negatively regulating cell growth. Heretofore, many studies have been conducted on genes contributing to carcinogenesis or a growth mechanism and peptides or proteins relating to them (Japanese Unexamined Patent Application Publication Nos. 2003-517306 and 2000-217585).

Among peptides or proteins derived from cell membrane surfaces of human cancer cells, generally, HLA-binding peptides are particularly thought to be cancer antigens and are thought to act on immunocompetent cells as antigens, most of all, as immunogens. Additionally, it is widely known that some proteins and glycoproteins derived from cancer cells and immunocompetent cells such as macrophages isolated from a cancer-bearing living body have an immunosuppressing activity preventing the destruction of cancer cells, for example, immunosuppressive acidic protein (IAP). Such proteins and glycoproteins are clinically used for measuring the degree of immunosuppression.

Heretofore, it has been thought that cancer cell growth progresses by a mechanism due only to self division of cancerous cells. However, the rate of cancer cell growth is not constant and cancer cells rapidly proliferate at some point. Such phenomena cannot be fully explained by the conventional view only.

SUMMARY OF THE INVENTION

On the basis of the above-mentioned phenomenon that cancer rapidly progresses at some point, cancer cells have a possibility of secreting a factor outside the cancer cells. Such a factor further accelerates the cancer cell growth by acting on normal cells neighboring the cancer cells so as to activate a cancer-related gene in the normal cells. If this factor is found, not only a mechanism of progression of cancer is clarified but also a useful drug can be provided.

Namely, it is an object of the present invention to provide a factor having a function of accelerating cancer cell growth by acting on normal cells neighboring the cancer cells so that a cancer-related gene in the normal cells is activated and to provide a corresponding gene of the factor. It is another object of the present invention to develop a valuable use of the factor and its corresponding gene by verifying the factor for the cancer-related-gene-activating function in normal cells.

The present inventor has performed intensive studies for overcoming the above-mentioned problems, and, as a result, found a peptide and a gene encoding the peptide in an extract of a human squamous-cell carcinoma cell line UTC-8 which has a high activity of metastasis. Furthermore, the inventor has proved that the peptide has a function of activating a cancer-related gene in normal cells neighboring cancer cells by acting on the normal cells and accelerating cancer growth, and has proved that the peptide can be used as an immunosuppressive agent. In addition, the inventor has found that a polynucleotide capable of hybridizing with the gene encoding the peptide can be used as a reagent for diagnosing malignancy of cancer or measuring tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration), which was not included in clinical examination items before, and that an antibody against the peptide has an anticancer activity. Thus, the present invention has been accomplished.

The present invention relates to aspects (1) to (10) described below:

(1) A peptide having an ability to activate a cancer-related gene, wherein the peptide is derived from cell membrane surfaces of human squamous-cell carcinoma cells and includes an amino acid sequence represented by SEQ ID No: 1 or an amino acid sequence having deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1;

(2) A peptide having an ability to activate a cancer-related gene, wherein the peptide is derived from cell line UTC-8 (FERM BP-08611) established from human squamous-cell carcinoma; and the peptide shows a detectable peak at a detection wavelength of 214 nm in gel filtration of an extract from the cell line;

(3) An immunosuppressive agent comprising the peptide according to the aspect (1) or (2) as an effective component;

(4) An antibody against the peptide according to the aspect (1) or (2);

(5) A diagnostic reagent including the antibody according to the aspect (4) for determining tendency of canceration or malignancy of cancer;

(6) An anticancer agent comprising the antibody according to the aspect (4) as an effective component;

(7) A polynucleotide including a nucleotide sequence encoding at least three contiguous amino acids of the amino acid sequence of the peptide according to the aspect (1);

(8) A diagnostic kit for determining tendency of canceration or malignancy of cancer, wherein the kit includes a peptide according to the aspect (1) or (2) and a polynucleotide according to the aspect (7);

(9) The diagnostic kit according to the aspect (8) further including at least one marker gene selected from the group consisting of:

Ras oncogene family;

v-crk avian sarcoma virus CT10 oncogene homolog-like lactate dehydrogenase B;

Placental growth factor;

Interleukin 8;

MAS1, activator of S phase kinase;

v-raf;

v-fms;

v-rel;

v-src;

GRO1;

Hepatoma-derived growth factor;

Vascular endothelial growth factor;

Bone morphogenic protein 3;

Squamous-cell carcinoma antigen recognized by T cell;

Interleukin-1 beta;

Conserved gene amplified in osteosarcoma; and

Lymphoid blast crisis oncogene; and

(10) A method for preparing a peptide according to the aspect (1) or (2), the method including the steps of immersing cells obtained by culturing human squamous-cell carcinoma cells in a culture medium into a citrate-phosphate buffer having a pH of 3.3 to 3.4 for extraction; and fractionating the extract by gel filtration using a citrate-phosphate buffer having a pH of 6.8 to 7.0 for yielding a peptide having an ability to activate a cancer-related gene in human normal cells.

The peptide according to the present invention is derived from cell membrane surfaces of human squamous-cell carcinoma cells. In particular, the peptide is extremely remarkable in the fact that it has ability to activate a cancer-related gene in human normal cells. Since the sensitivity of cells and tissues against this peptide of the present invention can be an indicator to predict canceration tendency of normal cells or progress of cancer, the peptide can be a useful diagnostic reagent for determining tendency of canceration or malignancy of cancer. Additionally, it is strongly suggested that the unregulated growth of cancer cells is caused by, not only cancer cells themselves, but also the interaction accompanying the activation of various cancer-related genes in normal cells. Thus, an entirely novel mechanism of cancer growth is presented.

Therefore, the present invention can contribute greatly to the research and development of diagnosis and therapy of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are elution patterns of gel filtration of peptides extracted in Example 1 according to the present invention. FIG. 1A shows elution patterns of the peptides by using a citrate-phosphate buffer having a pH of 7.2 as an elution buffer and detection wavelengths of 280 nm and 210 nm. FIG. 1B shows elution patterns of the fraction (elution volume: 21 ml) of the rightmost peak of the elution pattern using the detection wavelength of 280 nm in FIG. 1A by using a citrate-phosphate buffer having a pH of 6.8 as an elution buffer and detection wavelengths of 280 nm and 214 nm.

FIGS. 2A and 2B are electrophoresis photographs showing gene pattern changes in cells treated with a peptide of the present invention which were investigated by conducting a PCR reaction using cDNA prepared by reverse transcription of total mRNA derived from the cells treated with the peptide as a template and using DNA encoding the full length of the peptide of the present invention as a primer.

FIGS. 3A and 3B are electrophoresis photographs showing gene pattern changes in cells treated with a peptide of the present invention. The gene was prepared by a PCR reaction using cDNA prepared by reverse transcription of total mRNA derived from the cells treated with the peptide as a template and using DNA encoding a sequence consisting of the first to seventh amino acids of the peptide of the present invention as a primer.

FIG. 4 is a photograph showing the result when the monoclonal antibody against the peptide of the present invention was subjected to a cytotoxic test using cancer cells.

FIG. 5 is a diagram showing an immunosuppressive activity of the peptide of the present invention in a transplantation test.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The peptide according to the present invention has a function of activating a human cancer-related gene and includes the following amino acid sequence:

(SEQ ID NO: 1) Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu-Ser-Lys

Additionally, a peptide including an amino acid sequence having deletion, substitution, or addition of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1 is included in the present invention as long as the peptide has the function of activating a cancer-related gene.

The above-mentioned cancer-related gene refers to not only oncogenes but also immune system genes. The activation of a cancer-related gene means acceleration of canceration or cancer growth through activation of an oncogene or through inactivation of a tumor suppressor gene and/or activation or inactivation of an immune system gene, e.g., a cellular immunity system and/or humoral immunity system.

The immunosuppressive activity of the peptide according to the present invention is notable. For example, it is recognized that the peptide suppresses the rejection in tissue transplantation. Therefore, the peptide of the present invention can be used as an immunosuppressive agent.

The peptide represented by SEQ ID NO: 1 is derived from cell membrane surfaces of human squamous-cell carcinoma cells and can be extracted from human squamous-cell carcinoma cell line UTC-8 (Deposition No.: FERM BP-08611, which is deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan, on Feb. 4, 2004) by using a citrate-phosphate buffer having a pH of 3.3 to 3.4.

More specifically, after the extraction by the citrate-phosphate buffer having a pH of 3.3 to 3.4, the extract is further applied to gel filtration using a citrate-phosphate buffer having a pH of 6.8 to 7.2 as an elution buffer at a column flow rate of 0.1 to 0.28 ml/min, and then a fraction having a peak in a detection wavelength of 214 nm is isolated to yield the peptide represented by SEQ ID NO: 1.

The peptide represented by SEQ ID NO: 1 of the present invention is characterized by the fact that the peak can be separated and detected only when a detection wavelength of 214 nm is used. The peak of the peptide of the present invention cannot be detected when a detection wavelength of 254 to 257 nm or of 280 nm, which are generally used in peptide detection, is used.

In addition to the above-mentioned extraction method, the peptide according to the present invention can be prepared by conventional chemical synthesis of peptides on the basis of the amino acid sequence. Namely, an amino acid derivative having a carboxyl group and a side-chain functional group being protected by a protecting group and an amino acid derivative having an amino group and a side-chain functional group being protected by a protecting group are condensed in the presence of carbodiimide or the like. Then, the protecting group for the amino group is removed to bind a next amino acid derivative protected by a protecting group. Such a reaction can be performed by a liquid-phase method or a solid-phase method. In general, the solid-phase method is used, except when a relatively large amount of peptide is synthesized. Particularly, in a method developed for identifying an antigenic determinant defined by its amino acid sequence, a spacer arm having an amino group on its tip is used. The spacer arm is prepared by chemically treating a cellulose film or the tip of a plastic pin. In the latter method, a peptide chain is extended by sequential reactions of solutions in a 96-well plate; this method is called multi-pin peptide synthesis.

Furthermore, in another method, a peptide of the present invention may be prepared by chemically synthesizing a DNA encoding the peptide by using a DNA synthesizer; preparing a recombinant vector by connecting the DNA to an appropriate expression vector; introducing the vector into a host such as Escherichia coli; culturing the host; and collecting the peptide of the present invention from the culture.

The peptide of the present invention is useful by itself and can be used as a diagnostic reagent for determining tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration) of normal cells or tissues, for determining whether or not a subject is suffering from cancer, and for determining malignancy of cancer.

The above-mentioned various determinations are performed by bringing the peptide of the present invention in contact with a human cell or tissue specimen obtained by surgical resection or biopsy, and utilizing this contact for detecting a cancer-related gene or for determining a change in the expression degree or the expression pattern of the gene. The sensitivity of cells or tissues against the peptide of the present invention reflects, for example, a tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration) of the cells or tissues or a degree of malignancy of cancer.

In the determination of the degree of malignancy of cancer cells or tissues, the amount of expression of the gene encoding the peptide of the present invention may be directly determined without the contact of the peptide of the present invention with the cells or tissues. However, it is desirable that the cancer cells or tissues are brought into contact with the peptide of the present invention, in order to achieve higher sensitivity in measurement.

Additionally, as is clear in diagnosis methods 1 and 2 described below, a polynucleotide (DNA and RNA) encoding at least three contiguous amino acids of the amino acid sequence of the peptide of the present invention can be used as a diagnostic reagent for determining tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration) of normal cells or tissues, for determining whether or not a subject is suffering from cancer, and for determining malignancy of cancer. A diagnostic kit of the present invention preferably utilizes such a polynucleotide in combination with a peptide of the present invention.

Diagnosis methods using the peptide of the present invention will now be described.

[Diagnosis Method 1]

First, a specimen such as cells and tissues obtained from a subject to be tested is brought into contact with the peptide of the present invention, and they are cultured for a predetermined period of time. Examples of the specimen include cells and tissues obtained from a high risk group for cancer, such as subjects living in area polluted with a chemical or radioactive material and smokers; cells and tissues which are suspected to be a precancerous stage, such as polyp; cells and tissues neighboring cancer cells; cells and tissues at a region under a danger of metastasis of cancer; and peripheral blood.

When the specimen has a tendency of canceration, even if the cells and tissues are normal now, a change in an expression pattern of the gene is observed: the gene encoding the peptide of the present invention is detected, or the amount of expression of the gene is increased. When the malignancy of cancer is high, the amount of expression of the gene is further increased. Therefore, the tendency of canceration or malignancy of cancer can be determined by detecting the gene or determining the degree of its expression.

In this diagnosis method, the cells or tissues of interest are brought into contact with the peptide, and then total mRNA is extracted from the cells or tissues. After synthesis of cDNA by reverse transcription using the mRNA as a template, PCR is further conducted by using the resulting cDNA as a template. In the latter PCR, all polynucleotides encoding sequences consisting of at least three contiguous amino acid residues of the amino acid sequence represented by SEQ ID NO: 1 are collectively used as primers. The polynucleotide may encode the full-length polypeptide.

This diagnosis method will now be specifically described.

First, total mRNA is extracted by a common method from a specimen which is prepared by treating normal tissues or cells with the peptide. Then, cDNA is synthesized by reverse transcription using the total mRNA as templates to generate a cDNA pool which will be used as templates.

DNA encoding a sequence consisting of, for example, the first to fourth amino acids in the amino acid sequence represented by SEQ ID NO: 1 has the following nucleotide sequence:

CAR CCN CAR TTY (SEQ ID NO:2) (wherein N is A, T, C, or G, R is G or A, and Y is C or T). Each of the nucleotide sequences, i.e., 32 types of DNA, is synthesized, and a pool including each DNA in an equal amount is used as forward primers.

Since the cDNA used as the template has a poly-T sequence corresponding to poly-A tailing of mRNA, an oligo-dT primer (poly-A primer) is used as a reverse primer.

The PCR using the cDNA pool as the templates is conducted by using the above-mentioned forward primers and the reverse primer, followed by electrophoresis and fluorescence staining.

When a change in an expression pattern of genes including the gene encoding the peptide of the present invention is induced by treating a specimen with the peptide of the present invention, it is indicated that the specimen is sensitive to the treatment with the peptide of the present invention; thus, it is determined that the specimen has a high tendency of canceration even if the cells or tissues as the specimen are currently normal. When a specimen is already cancerous, a change in the gene expression pattern increases with a degree of the malignancy. Therefore, the degree of the malignancy can be determined by measuring the change.

The amount of the peptide expression can be determined by measuring the fluorescence intensity of the band in the electrophoresis. In order to precisely determine the amount of the peptide expression, for example, quantitative PCR may be performed.

The quantitative PCR can be performed by, for example, real-time PCR using the cDNA pool as templates, the above-mentioned primers, and a fluorescent dye or a fluorescence-labeled probe which binds with double-strand DNA. Then, a relationship between the number of the PCR cycles and the fluorescence intensity is determined. By comparing the results with those obtained by using a standard, the amount of the cDNA, i.e., the expression amount of the peptide of the present invention, can be quantitatively determined.

A change in the gene expression pattern or an increase in the peptide expression amount can be determined by comparing the expression level of a control. As the control, the results obtained by conducting the same procedure as above using the same cells except that the peptide of the present invention is not used and/or the results obtained by treating normal cells or tissues that are completely free from cancer risk with the peptide and conducting the same procedure as above, are used.

The normal cells and tissues that are free from cancer risk are derived from young subjects. As such cells, for example, a kidney mesangium cell (ACBRI-1376; Applied Cell Biology Research Institute), a skin fibroblast cell (Catlog2F0-C25; Cell Systems), and a pancreatic epithelial cell (CBRI515; Applied Cell Biology Research Institute) are commercially available.

From the views described above, it is obvious that a combination of the peptide of the present invention and the group of the above-mentioned primers, i.e., the group of polynucleotides encoding sequences consisting of at least three contiguous amino acid residues of the amino acid sequence represented by SEQ ID NO: 1, is useful as a cancer diagnostic reagent kit for determining a tendency of canceration or malignancy of cancer.

[Diagnosis Method 2]

In another diagnosis method using the peptide of the present invention, cancer-related gene is used as a marker gene.

In this diagnosis method, cells or tissues of interest are treated with the peptide of the present invention as in the above-mentioned diagnosis method 1; thus the same specimen as in the diagnosis method 1 can be used. However, in this diagnosis method, the reverse transcription is conducted to generate a cDNA (first-strand cDNA) pool, which is complementary to the mRNA, by using total mRNA extracted from the cells or tissues treated with the peptide as a template and using oligo-dT primer having T7 RNA polymerase promoter region as a reverse primer. Then, after the second-strand cDNA synthesis, in vitro transcription (IVT) was conducted using T7 RNA polymerase and biotinylated rNTPs at 37° C. for 14 hr to yield biotin-labeled cRNA. The amount of the cRNA is measured with a spectrophotometer to confirm that at least 10 μL of cRNA can be yielded.

The diagnosis method further includes processes for bringing this cRNA labeled with fluorescence dye or the like into contact with cDNA of a cancer-related gene which is immobilized on a plate for hybridization and for measuring the fluorescence intensity.

When the cRNA is hybridized to some cancer-related gene, it is suggested that the cancer-related gene is being expressed in the cells or tissues. When the fluorescence intensity of some cancer-related gene is increased or decreased, it is suggested that the expression amount of the cancer-related gene is increased or decreased by the treatment with the peptide of the present invention. Namely, for example, when the expression of an oncogene is increased or the expression of an immune system gene which suppresses canceration is decreased, it is suggested that the tendency of canceration is high. Thus, on the basis of the expression condition of a cancer-related gene, a tendency of canceration or malignancy of cancer can be determined.

An increase or decrease in the expression amount of a cancer-related gene is determined by comparing the expression level in a control. The cells and tissues used as the control are the same as those used in the diagnosis method 1.

As described above, immune system genes relating to cancer, in addition to oncogenes, are included in the cancer-related genes in this specification. Examples of such genes are as follows:

Ras oncogene family;

v-crk avian sarcoma virus CT10 oncogene homolog-like lactate dehydrogenase B;

Placental growth factor;

Interleukin 8;

MAS1, activator of S phase kinase;

v-raf;

v-fms;

v-rel;

v-src;

GRO1;

Hepatoma-derived growth factor;

Vascular endothelial growth factor;

Bone morphogenic protein 3;

Squamous-cell carcinoma antigen recognized by T cell;

Interleukin-1 beta;

Conserved gene amplified in osteosarcoma; and

Lymphoid blast crisis oncogene.

At least one gene of these cancer-related genes is used in this invention. For higher precision, yet more cancer-related genes may be used. In such a case, it is preferable to use a combination of genes in the following groups as a genes set for diagnosis.

(a) Oncogene expression-increasing group: a gene encoding the peptide of the present invention and Ras oncogene and c-fos oncogene, etc.;

(b) Immune gene expression-increasing group: Interleukin-1 beta, etc.; and

(c) Immune gene expression-decreasing group: MHC class II, DM, and alpha, beta protein gene and killer cell lectin-like receptor subfamily B, M member 1 gene.

Tables 1 and 2 show examples of cancer-related genes of which expression is increased and immune system genes of which expression is increased or decreased by the treatment with the peptide (HPLC-purified peptide) derived from cell membrane surfaces of human squamous-cell carcinoma cells UTC-8 (Deposition No.: FERM BP-08611, which is deposited with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology).

TABLE 1 Cancer-related gene enhanced by the HPLC-purified peptide Gene Name Fold Change Common Description Product Phenotype Function Keywords K03218 3.912448639 SRC; v-src proto-oncogene Colon c-myc ASV; sarcoma tyrosine-protein cancer, proto- SRC1; (Schmidt- kinase SRC advanced oncogene; c-SRC; Ruppin A-2) proto- p60-Src viral oncogene; oncogene src gene; homolog src (avian) oncogene NM_001201 3.73617959 BMP3 bone bone morphogenetic morphogenetic protein 3 (osteogenic) protein 3 precursor (osteogenic) D16431 3.595973492 HDGF hepatoma- hepatoma-derived hepatoma- derived growth factor (high- derived growth mobility group protein GF; factor 1-like) hepatoma- (high- derived mobility growth group factor protein 1- like) X54936 3.576227427 PGF placental placental growth placenta growth factor, vascular growth factor, endothelial growth factor vascular factor-related protein endothelial growth factor- related protein M95712 3.033826351 BRAF v-raf murine v-raf murine sarcoma Adenocarcinoma b-raf sarcoma viral oncogene homolog of oncogene; viral B1 lung, serine/threonine oncogene somatic; protein homolog B1 Colorectal kinase cancer, somatic; Melanoma, melignant, somatic; Nonsmall cell lung cancer, somatic NM_005850 3.023255825 SF3B4 splicing splicing factor 3b, factor 3b, subunit 4 subunit 4, 49 kDa NM_005937 2.995664358 MLLT6 myeloid/lymphoid myeloid/lymphoid or or mixed-lineage leukemia mixed- (trithorax homolog, lineage Drosophila); leukemia translocated to, 6 (trithorax homolog, Drosophila); translocated to, 6 NM_001419 2.950166225 ELAVL1 ELAV ELAV-like 1 (embryonic lethal, abnormal vision, Drosophila)- like 1 (Hu antigen R) NM_002613 2.86612194 PDPK1 3- 3-phosphoinositide protein phosphoinositide dependent protein kinase dependent kinase-1 protein kinase-1 NM_014308 2.849243879 P101- phosphoinositide- phosphoinositide-3- PI3K 3- kinase, regulatory kinase, subunit, polypeptide regulatory p101 subunit, polypeptide p101 D26120 2.833239079 splicing factor 1 X61498 2.832505941 NFKB2 nuclear nuclear factor of NF-kb factor of kappa light subunit kappa light polypeptide gene polypeptide enhancer in B-cells 2 gene (p49/p100) enhancer in B-cells 2 (p49/p100) X03663 2.780905962 CSF1R colony colony stimulating Myeloid c-fms stimulating factor 1 receptor malignancy, oncogene; factor 1 precursor predisposition fms receptor, to oncogene; formerly glycoprotein; McDonough membrane feline protein; sarcoma proto- viral (v- oncogene; fms) signal oncogene peptide homolog NM_004204 2.756244421 PIGQ phosphatidylinositol phosphatidylinositol glycan, glycan, class Q class Q isoform 2; phosphatidylinositol glycan, class Q isoform 1 NM_003017 2.741738006 SFRS3 splicing splicing factor, factor, arginine/serine-rich 3 arginine/serine- rich 3 NM_000753 2.7408638 PDE3B phosphodiesterase phosphodiesterase 3B, 3B, cGMP-inhibited cGMP- inhibited NM_002712 2.696629047 PPP1R7 protein protein phosphatase 1, regulatory phosphatase regulatory subunit 7 polypeptide 1, of regulatory protein subunit 7 phosphatase-1 NM_004906 2.543901205 WTAP Wilms tumor Wilms' tumour 1- 1 associated associating protein protein isoform 1; Wilms' tumour 1-associating protein isoform 2 NM_005207 2.497231245 CRKL v-crk v-crk sarcoma virus sarcoma CT10 oncogene homolog virus CT10 (avian)-like oncogene homolog (avian)-like NM_007279 2.493814707 U2AF2 U2 small U2 small nuclear nuclear ribonucleoprotein ribonucleoprotein auxiliary factor auxiliary (65 kD) factor (65 kD) NM_015714 2.480620146 G0S2 putative putative lymphocyte lymphocyte G0/G1 switch gene G0/G1 switch gene NM_001511 2.476593256 CXCL1 chemokine chemokine (C—X—C (C—X—C motif) ligand 1 motif) ligand 1 (melanoma growth stimulating activity, alpha) NM_016263 2.465854406 FZR1 Fzr1 protein Fzr1 protein NM_012103 2.459948301 AUP1 ancient ancient ubiquitous ubiquitous protein 1 isoform 1; protein 1 ancient ubiquitous protein 1 isoform 2; ancient ubiquitous protein 1 isoform 3 M32977 2.456767944 VEGF vascular vascular endothelial Diabetic angiogenic endothelial growth factor retinopathy, mitogen; growth NIDDM- vascular factor related, endothelial susceptibility growth to factor NM_007040 2.432915688 E1B-AP5 E1B-55 kDa- E1B-55 kDa-associated associated protein 5 isoform a; protein 5 E1B-55 kDa-associated protein 5 isoform d; E1B-55 kDa-associated protein 5 isoform b; E1B-55 kDa-associated protein 5 isoform c NM_001402 2.404632807 EEF1A1 eukaryotic eukaryotic translation translation elongation factor 1 elongation alpha 1 factor 1 alpha 1 NM_002357 2.396934271 MAD MAX MAX dimerization dimerization protein 1 protein 1 NM_005524 2.39485383 HES1 hairy and hairy and enhancer of enhancer of split 1 split 1, (Drosophila) NM_005730 2.387011766 CTDSP2 conserved nuclear LIM interactor- gene interacting factor 2 amplified in osteosarcoma AF040963 2.356589317 MXD4 MAX MAD4 dimerization protein 4 M83221 2.351067066 RELB v-rel reticuloendotheliosis NF-kappa-B I-Rel; reticuloendotheliosis viral oncogene homolog B transcpription NF-kappa-B viral factor transcription oncogene p50- factor homolog B, subunit inhibitor nuclear inhibitor factor of kappa light polypeptide gene enhancer in B-cells 3 (avian) NM_006842 2.24469161 splicing factor 3b, subunit 2, 145 kDa NM_001570 2.239902258 IRAK2 interleukin-1 interleukin-1 receptor- receptor- associated kinase 2 associated kinase 2 NM_006716 2.230443954 ASK activator activator of S phase of S phase kinase kinase NM_004723 2.209302187 ARHGEF2 rho/rac rho/rac guanine guanine nucleotide exchange nucleotide factor 2 exchange factor (GEF) 2 M60119 2.204995632 HIVEP2; human human immunodeficiency MBP-2; immunodeficiency virus type I enhancer HIV-EP2 virus binding protein 2 type I enhancer binding protein 2 NM_001665 2.111301422 ARHG ras homolog ras homolog gene gene family, member G (rho family, G) member G (rho G) M62829 2.104651213 EGR1 early early growth response 1 transcription growth factor response 1 NM_004218 2.102017164 RAB11B RAB11B, RAB11B, member RAS member RAS oncogene family oncogene family NM_006318 2.098191261 putative glialblastoma cell differentiation- related U33819 2.0930233 MAZ MYC- MYC-associated zinc associated finger protein zinc finger protein (purine- binding transcription factor) M13150 2.069767475 MAS1 MAS1 MAS1 oncogene mas oncogene oncogene; mas protein; membrane protein; proto- oncogene NM_003685 2.039983511 KHSRP KH-type KH-type splicing splicing regulatory protein regulatory (FUSE binding protein protein 2) (FUSE binding protein 2) NM_006694 2.018802404 JTB; jumping jumping translocation PAR; translocation breakpoint hJT; breakpoint HJTB; B PAR NM_006191 2.002365112 PA2G4 proliferation- proliferation- associated associated 2G4, 38 kDa 2G4, 38 kDa X78710 2.001409531 MTF1 metal- metal-regulatory metal- regulatory transcription factor 1 regulatory transcription transcription factor 1 factor; MTF-1 gene; transcription factor

TABLE 2 Immune system gene regulated by the HPLC-purified peptide Gene Name TDP Description Product Phenotype function Keywords Immune system gene enhanced by the peptide 1 K02770 11.69 interleukin 1, beta interleukin 1, beta interleukin- interleukin; proprotein 1 receptor interleukin 1 binding; signal transducer activity 2 J04130 7.341 chemokine (C-C motif) chemokine (C-C receptor act2 ligand 4 motif) ligand 4 signaling gene; precursor protein immune tyrosine activation kinase gene activity; chemokine activity 3 NM_000211 3.326 integrin, beta 2 integrin beta chain, Leukocyte cell (antigen CD18 (p95), beta 2 precursor adhesion adhesion lymphocyte function- deficiency receptor associated antigen 1; activity macrophage antigen 1 (mac-1) beta subunit) 4 X03663 2.781 colony stimulating colony stimulating Myeloid macrophage c-fms factor 1 receptor, factor 1 receptor malignancy, colony oncogene; formerly McDonough precursor predisposition stimulating fms feline sarcoma viral to factor oncogene; (v-fms) oncogene receptor glycoprotein; homolog activity; membrane ATP binding; protein; transferase proto- activity oncogene; signal peptide 5 NM_006186 2.06 nuclear receptor nuclear receptor Parkinson steroid subfamily 4, group A, subfamily 4, group disease hormone member 2 A, member 2 isoform receptor a; nuclear receptor activity; subfamily 4, group transcription A, member 2 isoform factor b; nuclear receptor activity subfamily 4, group A, member 2 isoform c; nuclear receptor subfamily 4, group A, member 2 isoform d 6 NM_002000 1.871 Fc fragment of IgA, Fc alpha receptor receptor receptor for isoform a precursor; activity; Fc alpha receptor receptor isoform b precursor; signaling Fc alpha receptor protein isoform c precursor; activity Fc alpha receptor isoform d; Fc alpha receptor isoform e; Fc alpha receptor isoform f; Fc alpha receptor isoform g; Fc alpha receptor isof 7 NM_003199 1.621 transcription factor 4 transcription factor RNA 4 isoform b polymerase II transcription factor activity; DNA binding 8 NM_001733 1.543 complement component complement component C1r/C1s complement 1, r subcomponent 1, r subcomponent deficiency, component combined C1r activity; trypsin activity; calcium ion binding; chymotrypsin activity; hydrolase activity 9 NM_002983 1.542 chemokine (C-C motif) chemokine (C-C chemokine ligand 3 motif) ligand 3 activity; antiviral response protein activity; signal transducer activity 10 NM_002969 1.53 mitogen-activated mitogen-activated MAP kinase protein kinase 12 protein kinase 12 activity; ATP binding; protein serine/threonine kinase activity; transferase activity; SAP kinase 3 activity 11 NM_000173 1.529 glycoprotein Ib platelet Bernard- thrombin (platelet), alpha glycoprotein Ib Soulier receptor polypeptide alpha polypeptide syndrome activity; precursor cell adhesion molecule activity 12 D10202 1.506 platelet-activating platelet-activating platelet G-protein factor receptor factor receptor activating coupled factor receptor; receptor PAF activity receptor; platelet- activating factor receptor Immune system gene suppressed by the peptide 1 NM_006864 0.662 leukocyte leukocyte receptor immunoglobulin-like immunoglobulin-like activity receptor, subfamily B receptor, subfamily (with TM and ITIM B (with TM and ITIM domains), member 3 domains), member 3 2 NM_007052 0.662 NADPH oxidase 1 NADPH oxidase 1 superoxide- isoform long; NADPH generating oxidase 1 isoform NADPH oxidase short; NADPH activity; oxidase 1 isoform oxidoreductase long variant activity; voltage-gated proton channel activity 3 NM_002258 0.653 killer cell lectin- killer cell lectin- sugar binding; like receptor like receptor transmembrane subfamily B, member 1 subfamily B, member 1 receptor activity 4 NM_001776 0.646 ectonucleoside ectonucleoside apyrase triphosphate triphosphate activity; diphosphohydrolase 1 diphosphohydrolase 1 magnesium ion binding; hydrolase activity 5 NM_016523 0.644 killer cell lectin- killer cell lectin- transmembrane like receptor like receptor receptor subfamily F, member 1 subfamily F, member 1 activity 6 NM_014442 0.641 sialic acid binding sialic acid binding sugar binding; Ig-like lectin 8 Ig-like lectin 8 transmembrane receptor activity; cell adhesion molecule activity 7 NM_004133 0.635 hepatocyte nuclear hepatocyte nuclear steroid factor 4, gamma factor 4, gamma hormone receptor activity; steroid binding; transcription factor activity 8 NM_002121 0.633 major major Beryllium class II major histocompatibility histocompatibility disease, histocompatibility complex, class II, DP complex, class II, chronic, complex beta 1 DP beta 1 precursor susceptibility antigen to 9 NM_000397 0.631 cytochrome b-245, cytochrome b-245, Chronic voltage-gated beta polypeptide beta polypeptide granulomatous ion channel (chronic (chronic disease, activity; granulomatous granulomatous X-linked electron disease) disease) transporter activity; oxidoreductase activity 10 NM_004750 0.626 cytokine receptor- cytokine receptor- Cold- receptor like factor 1 like factor 1 induced activity sweating syndrome 11 NM_003891 0.618 protein Z, vitamin K- protein Z, vitamin trypsin dependent plasma K-dependent plasma activity; glycoprotein glycoprotein protein binding; calcium ion binding; chymotrypsin activity 12 NM_002260 0.603 killer cell lectin- killer cell lectin- sugar binding; like receptor like receptor transmembrane subfamily C, member 2 subfamily C, member 2 receptor activity 13 NM_005545 0.579 immunoglobulin immunoglobulin protein superfamily superfamily binding containing leucine- containing leucine- rich repeat rich repeat 14 NM_001311 0.561 cysteine-rich protein cysteine-rich zinc ion 1 (intestinal) protein 1 binding (intestinal) 15 NM_004528 0.561 microsomal microsomal peroxidase glutathione S- glutathione S- activity; transferase 3 transferase 3 glutathione transferase activity 16 NM_018661 0.528 defensin, beta 103 defensin, beta 103, antimicrobial precursor peptide activity; Gram-positive antibacterial peptide activity 17 NM_002124 0.502 major major Pemphigoid, MHC class II histocompatibility histocompatibility susceptibility receptor complex, class II, DR complex, class II, to activity beta 1 DR beta 1 precursor 18 Y00815 0.475 protein tyrosine protein tyrosine antigen; phosphatase, receptor phosphatase, cell type, F receptor type, F surface isoform 1 glycoprotein; precursor; protein glycoprotein; tyrosine immunoglobulin phosphatase, superfamily; receptor type, F LAR isoform 2 precursor gene; leukocyte common antigen; neural cell adhesion molecule; transmembrane protein 19 NM_004636 0.459 sema domain, sema domain, immunoglobulin domain immunoglobulin (Ig), short basic domain (Ig), short domain, secreted, basic domain, (semaphorin) 3B secreted, (semaphorin) 3B 20 NM_001783 0.4 CD79A antigen CD79A antigen transmembrane (immunoglobulin- isoform 1 receptor associated alpha) precursor; CD79A activity antigen isoform 2 precursor 21 NM_004106 0.382 Fc fragment of IgE, Fc fragment of IgE, receptor high affinity I, high affinity I, signaling receptor for; gamma receptor for; gamma protein polypeptide polypeptide activity; precursor transmembrane receptor activity; IgE binding 22 U77604 0.349 microsomal microsomal glutathione glutathione S- glutathione S- transferase transferase 2 transferase 2 activity; enzyme activator activity 23 NM_002118 0.335 major major chaperone histocompatibility histocompatibility activity; MHC complex, class II, DM complex, class II, class II beta DM beta precursor receptor activity 24 NM_002123 0.328 major major Creutzfel histocompatibility histocompatibility dt-Jakob complex, class II, DQ complex, class II, disease, beta 1 DQ beta 1 precursor variant, resistance to 25 K01171 0.319 major major MHC class II antigen; histocompatibility histocompatibility receptor class II complex, class II, DR complex, class II, activity antigen; alpha DR alpha precursor histocompatibility antigen; major histocompatibility complex 26 NM_006120 0.298 major major histocompatibility histocompatibility complex, class II, DM complex, class II, alpha DM alpha precursor

The present inventor has verified the validity of this diagnosis method using 55000 cancer-related genes. When such a great number of cancer-related genes are used, DNA chip technology is preferably used. In the DNA chip technology, each DNA of the cancer-related genes is immobilized so as to be arrayed on a slide; the above-mentioned labeled cRNA solution is dropped into the slide; the cRNA which is not hybridized is removed by washing; and fluorescence intensity of each gene position is detected by a scanner. The results are compared with the result which is obtained by conducting the same process using the above-mentioned control solution. Thus, cancer-related genes of which expression is increased or decreased when cells or tissues are treated with the peptide of the present invention can be exhaustively analyzed.

Therefore, a combination of such a cancer-related gene, the peptide of the present invention, and a polynucleotide encoding the peptide is useful as a cancer diagnostic reagent kit for determining a tendency of canceration or malignancy of cancer.

[Diagnosis Method 3]

The peptide of the present invention is also used as an antigen for preparing a specific antibody against the peptide. The specific antibody is useful as a diagnostic reagent. The antibody may be either polyclonal or monoclonal antibody, and these antibodies can be prepared by known methods. For example, the polyclonal antibody can be prepared as an antiserum by immunizing an animal such as mouse, rat, or rabbit with the peptide of the present invention. The antiserum may be further purified. The monoclonal antibody can be prepared by a hybridoma method: extracting spleen cells from the above-mentioned sensitized animal and fusing the spleen cells with myeloma cells to form hybridomas.

In the immunization, an appropriate adjuvant may be used. In addition, the peptide of the present invention may be used as a conjugate with a carrier protein such as keyhole limpet hemocyanin for increasing the immunogenicity of the peptide.

The antibody against the peptide of the present invention is used as a diagnostic reagent for determining malignancy of cancer, wherein a specimen, such as cancer tissues or cells, is stained by using the specific antibody labeled with an appropriate fluorescence dye or the like. When the peptide of the present invention is detected in the tissues or cells, it is suggested that cancer is advancing. The amount of the peptide can be determined by measuring the fluorescence intensity. A large amount of the peptide indicates that the malignancy of cancer is considerably high.

Additionally, the antibody against the peptide of the present invention can be used as a diagnostic reagent for measuring a tendency of canceration (easiness of conversion to cancer: a degree of risk of canceration). In such a use of the antibody, a specimen of the tissues or cells shown in diagnosis methods 1 and 2 is stained using the labeled antibody. When the peptide of the present invention is detected in these tissues or cells, it is suggested that the tissues or cells are exposed to cancer-inducing stress, i.e., a degree of risk of canceration is high. When the amount of the peptide of the present invention is large, it is suggested that the tendency is further higher. When the specimen is cancer cells, a large amount of the peptide suggests that the malignancy of the cancer is high.

Furthermore, the antibody against the peptide of the present invention can be expected to have an anticancer effect such as suppression of cancer cell proliferation as a cancer-cell proliferation-suppressing drug. In addition, the antibody can be expected to block malignant alteration or mutation acceleration to which normal cells are subjected as a cancer-preventing drug. Furthermore, by binding the antibody to an anticancer agent, a drug which may be used for missile therapy can be obtained.

EXAMPLES

Examples of the present invention will now be described, but the present invention is not limited to these examples.

Example 1 Preparation and Sequencing of the Peptide of the Present Invention

(Cell: UTC-8)

The squamous-cell carcinoma cell (UTC-8: FERM BP-08611) is a highly differentiated type with high metastasis potential and keratinization tendency. The cell also has high adhesion and proliferation ability and becomes confluent in 5 to 7 days after inoculating 1×10⁵ cells into a culture medium (5 ml) in a T25 culture flask (Falcon).

(Cell-Culturing Condition)

The following processes were performed: inoculating 1×10⁶ UTC-8 cells into 10 T150 culture flasks (Falcon); performing initial culture in 20 ml RPMI1640 culture medium with 10% FCS (GIBCO); and additionally supplying 30 ml of the culture medium when the proliferation reached about 30% of the culture flask dimension. The culture was performed in 5% CO₂ and 95% humidity at an incubator internal temperature of 37° C. When the cells become 80% confluent (the proliferation of the cells is maintained at the point just before the cell proliferation reaches its peak), the antigen peptide bound to HLA was eluted from the cell membrane surfaces.

(Antigen Peptide-Eluting Procedure)

The culture medium for the UTC-8 cells was removed when the cells became 80% confluent in each T150 culture flask (Falcon). The cells were washed with Hanks solution once and then with 30 ml of a PBS solution not containing divalent calcium ion and divalent magnesium ion twice. Then, after the sufficient removal of the solution, 10 ml of a citrate-phosphate buffer having a pH of 3.3 to 3.4 was added to each flask, and the cells were left at a room temperature for 2 min.

Then, the citrate-phosphate buffer solution was collected and centrifuged at 1200 rpm for 7 min, and the supernatant was filtered through a 0.45 μm filter (Millex-HV PVDF: MILLIPORE). The filtrate was further filtered through a 0.22 μm filter. The filtrate was desalted with Sep-Pac C18 cartridge (Waters), and the target substance bound to the cartridge was eluted with a 60% (v/v) acetonitrile aqueous solution as a crude extract solution. The thus obtained crude extract solution was frozen and stored at −20° C. for later HPLC.

The cells received the above-described treatment were washed, immediately after the collection with the citrate-phosphate buffer solution having a pH of 3.3 to 3.4, with 30 ml of Hanks solution twice. Then, the cells were recultured in 30 ml of RPMI 164 medium containing 10% FCS. This treatment was subjected to the same cells once a day for successive 4 days.

(Lyophilization and Redissolution of Crude Extraction)

The crude extract solution obtained in above was lyophilized using a lyophilizer (FD-1000: EYELA) under conditions at a trapping temperature of −40° C. and at a degree of vacuum of 15 Pa or less. The dried sample was redissolved in 5 ml of the citrate-phosphate buffer solution having a pH of 3.3 to 3.4. At this stage, the total protein amount determined by Lowry method was 400 μg.

(Two-Dimensional Electrophoresis)

Ten micrograms of the thus obtained molecule was applied to two-dimensional electrophoresis, but no band was visible to the naked eye by Coomassie Brilliant Blue (CBB) R250 staining (detection sensitivity: 1 μg) and also by silver staining having a sensitivity as high as about 1000 times that of the CBB staining, (theoretical sensitivity: 1 ng, actual sensitivity: about 20 times that of the CBB staining). This suggests that the peptide molecule physically passes through a gel (silica gel C18) generally used in the two-dimensional electrophoresis. This is an unusual characteristic of the peptide molecule of the present invention.

(Fractionating by Gel Filtration)

Since the molecule of the present invention cannot be detected by usual two-dimensional electrophoresis, it was tried to detect the molecule by an HPLC system. The target peptide was obtained as a fraction obtained by using an AKTA Explorer 10 (Amersham Pharmacia Biotech) HPLC system and Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech) as a gel-filtration column; using a citrate-phosphate buffer solution (0.1 M citric acid and 0.2 M phosphoric acid) having a pH of 6.8 as an elution buffer at a flow rate of 0.2 ml/min; and isolating a peak in a fraction at an elution volume of 20.8 to 22.8 ml by using a detection wavelength of 214 nm. The obtained fraction was lyophilized and then redissolved in sterilized redistilled water for desalting. The desalting was performed by fractionating by the AKTA Explorer 10 (Amersham Pharmacia Biotech) HPLC system and Superdex Peptide 10/300 GL (Amersham Pharmacia Biotech) as a gel-filtration column using distilled water as a solvent at a flow rate of 0.2 ml/min. The target peptide was obtained as a fraction at an elution volume of 11.4 to 20.0 ml.

FIG. 1 shows an elution profile of the gel filtration.

The peptide was lyophilized using the lyophilizer FD-1000 (EYELA) and stored at −20° C. for later mass spectrometry.

(Optimal pH)

The optimal pH of the elution buffer was 6.8 to 7.2. When the pH of the elution buffer was 7.3 or more, peaks were further divided into smaller peaks. When the pH of the elution buffer was 6.7 or less, peaks, which were isolated from each other at a pH of 6.8, lapped over each other, and the gel filtration resolution was decreased.

(Detection Wavelength)

The peptide is characterized by the fact that the peak of the peptide in the crude extract solution can be separately detected by using physiological activity as an indicator only when a detection wavelength of 214 nm is used. However, the peptide cannot be detected when a detection wavelength of 254 to 257 nm or of 280 nm, which are generally used in peptide detection, is used. These results suggest that the amino acid composition of the peptide has extremely low contents of tyrosine (absorption wavelength: 280 nm), tryptophan (absorption wavelength: 280 nm), and phenylalanine (absorption wavelength: 257 nm); or that the peptide does not substantially contain such amino acids. The use of a detection wavelength of 214 nm has a demerit such that many interfering substances are also detected by ultraviolet absorption spectrometry. However, it can precisely measure a protein amount (quantitative detection range: 5 to 1000 μg) regardless of types of proteins. The 214 nm is the most appropriate wavelength for, as in this experiment, precisely measuring a concentration of unknown peptide in a crude extract solution by detecting a peak utilizing peptide-bond absorption without denaturation of the peptide to maintain the physiological activity. On the other hand, the quantitative detection property was further improved by using a detection wavelength of 205 nm when the crude extract solution containing the peptide was applied to a desalting column (Sep-Pack 18) or to a molecular sieve (Centricon YM-3) for removing foreign substances such as nucleic acids as far as possible, by intending only purification of the peptide without any concern for maintaining the physiological activity. Therefore, when a peak in the crude extract solution was detected using the physiological activity as an indicator, a detection wavelength of 214 nm was used, and when the precise quantitative determination was necessary, a detection wavelength of 205 nm was used.

(Fractionating Flow Rate in Column)

The fractionating flow rate is important for separating the peptide. Namely, the flow rate of an elution buffer is determined so that molecules slowly pass through the column to effectively utilize advantages of the molecular sieve. When the flow rate was 0.28 ml/min, adjacent peaks lapped over each other; thus, the separation resolution was insufficient. For the detection of the peak of the present invention, a low flow rate of 0.14 ml/min was optimal. Each fraction was determined to be 0.1 ml/well from the view point of the width of the peak, and was collected in a 96-well plate (Nunc).

(Structure Analysis of the Peptide by Mass Spectrometer)

The peptide fraction that was stored after lyophilization was redissolved in sterilized redistilled water, and 5 μg as a protein was separated by ProteinChip Series 4000 system (Ciphergen). The sample (5 μg) was bound to the ProteinChip by using MilliQ water as a binding/wash buffer in normal-phase ProteinChip and using 100 mM sodium acetate (pH 4.0) as a binding/wash buffer in cation exchange ProteinChip, and was measured by using alpha-cyano-4-hydroxy-cinnamic acid (CHCA) as an energy-absorbing molecular in a mass range of 800 to 2500 m/z. As a result, a peak of a molecular weight which was thought to be that of the target peptide was detected. By using the detection conditions clarified by the above-described processes as a reference, the target peptide trapped on the chip was analyzed by a mass spectrometer, QSTAR XL LC/MC/MS system (Applied Biosystems). Then, on the basis of the results of time-of-flight (TOF mass spectrometry) analysis of the fragmented peptide-constituting portion, a data base (Mascot Search Results: Matrix Science) was searched for a sequence of the peptide to reveal that the peptide had the following peptide sequence of which function was unknown as of Jan. 27, 2005.

Peptide: (SEQ ID NO: 1) Gln-Pro-Gln-Phe-Gly-Arg-Arg-Met-Glu-Ser-Lys

Example 2 Confirmation of Expression of the Peptide of the Present Invention in Original Cancer Cell UTC-8

The fact that the peptide obtained in Example 1 was actually expressed in the original cancer cell UTC-8 at a gene level was confirmed as follows:

UTC-8 cells after the extraction of the peptide of the present invention were recultured, and total RNA was extracted from the recultured UTC-8 cells after 2 hr, 4 hr, and 6 hr from the extraction, respectively, by using an RNA extraction kit (QIAGEN). Then, cDNA was synthesized using each of the extracted total RNA as a template to produce a cDNA pool. On the basis of the amino acid sequence (SEQ ID NO: 1) of the peptide, the following primers were designed. Fw primers (DNAs encoding four contiguous amino acids of the peptide were synthesized so that the N-terminal of the four contiguous amino acids shifted one by one from the N-terminal of the peptide toward the C-terminal.)

MHC 1-4 CAR CCN CAR TTY (SEQ ID NO: 2) MHC 2-5 CCN CAR TTY GGN (SEQ ID NO: 3) MHC 3-6 CAR TTY GGN AGR (SEQ ID NO: 4) MHC 4-7 TTY GGN AGR AGR (SEQ ID NO: 5) MHC 5-8 GGN AGR AGR ATG (SEQ ID NO: 6) MHC 6-9 AGR AGR ATG GAR (SEQ ID NO: 7) MHC 7-10 AGR ATG GAR TCN (SEQ ID NO: 8) MHC 8-11 ATG GAR TCN AAR (SEQ ID NO: 9) (N: A, T, C, or G, R: G or A, Y: C or T) Rv primer (a gene sequence produced for an amino acid sequence at the C-terminal of the peptide): Oligo-dT primer (poly-A primer)

Here, the poly-A primer was used as the Rv primer in order to obtain cDNA synthesized from mRNA.

Complementary DNAs were synthesized by reverse transcription from total RNA which was extracted at each time to produce a cDNA pool. Then, PCR was performed using this cDNA pool as templates and using primers synthesized as described above under conditions at 94° C. for 3 min, at 55° C. for 1 min, and at 74° C. for 1 min as one cycle. The cycle was repeated 35 cycles in total. After agarose-gel electrophoresis, 12 bands were selected from a sample of which RNA was extracted after 4 hr when the signal intensity was largest. The size of each band was about 500 to 2000 bp. Genes of these bands extracted from the gel were inserted into pGM easy vectors.

Insert of the genes into 12 types of the pGM easy vectors was checked, and 8 samples per one type of the vector were sequenced. As a result, it was confirmed that all the sequences of the 8 samples included a common sequence shown in Table 3 below, though the DNA lengths of the sequences were different from those of each other.

TABLE 31

The result shows the fact that a gene being common in or complementary to the gene encoding the peptide of the present invention is surely present in original cancer cell UTC-8 and that the peptide is derived from UTC-8 cells.

Example 3 Artificial Synthesis of the Peptide of the Present Invention by Fmoc Method

The peptide was artificially synthesized by an in vitro synthesis system (Abacus: Sigma Genosys).

The peptide was synthesized using an activated cellulose membrane (SPOTs: Sigma Genosys) as follows: The α-amino groups of amino acids were protected with 9-fluorenyl methoxy carbonyl (Fmoc) and the carboxyl groups were protected with an active ester (Opfp or Odhbt). These amino acid derivatives were dissolved in 1-methyl-2-pyrrolidinone (NMP). About 1 μl of the amino acid derivative solution (equivalent to 100 to 200 μg of Fmoc-amino acid) was spotted on the membrane at each marked position where a spacer arm was bound thereto. The end of the spacer arm had a free amino group, and the spots were colored to blue with bromophenol blue (BPB). An amido bond was formed by the reaction between the amino group on the membrane and the active ester. Then, capping was carried out as follows: Excess amino acid derivatives were rinsed away with dimethylformamide (DMF), and then unreacted amino group was acetylated by treating with acetic anhydride/DMF so as to be lost the reactivity. Then, the Fmoc group protecting the amino group was removed by secondary amine, piperidine/DMF, in order to let the amino group free for the subsequent extension reaction. Additionally, after all synthesis processes were completed, deprotection of the side chains was carried out. Namely, a t-butyl alcohol-based protecting group (Pmc, OtBu, Trt, tBoc, tBu, etc.) protecting the reactive side chains was removed by using trifluoroacetate (TFA) mixed with dichloromethane (DCM)/triisobutylsilane. Thus, the target peptide was artificially synthesized.

Example 4 Influence of the Artificially Synthesized Peptide on Normal Cell Gene

(a) The peptide obtained in Example 3 was added to a human normal peripheral blood monocyte culture system (10 to 100 μg peptide/1×10⁶ to 1×10⁷ monocytes), and they were cocultured for 7 hr. Then, mRNA was extracted from the monocytes and was applied to analysis for up-regulation and down-regulation in respect to about 55000 human genes by using DNA chip system (Clontech). Table 4 shows a part of the genes.

TABLE 4 A. Enhanced Gene Expression Expression Amount Amount before after Treatment Treatment with with Gene Gene the the No. Gene Name Bank Peptide Peptide 1. Oncogene 1 21713 NM_004339.2 GE480864 DISCOVERY 0.168670654 7.741943359 2 32050 CB999164.1 GE540894 DISCOVERY 0.344818115 9.805541992 3 34854 NM_198502.1 GE603511 DISCOVERY 0.040802002 1.098205566 4 46997 AL831827.1 GE547122 DISCOVERY 11.17044067 236.1818237 5 37781 AI478531.1 GE645872 DISCOVERY 2.533325195 32.40939331 6 7394 AA021565.1 GE707587 DISCOVERY 29.597229 350.270813 7 22388 CD244420.1 GE692174 DISCOVERY 60.32098389 571.2325439 8 6231 BF973345.1 GE54360 DISCOVERY 0.583343506 5.453491211 9 34979 BC041456.1 GE631189 DISCOVERY 2.651672363 24.171875 10 4210 NM_152577.1 GE493259 DISCOVERY 61.90475464 393.7416382 11 36631 AW302705.1 GE53805 DISCOVERY 71.11248779 417.5 12 29317 AA601191.1 GE551636 DISCOVERY 2.891571045 10.05682373 13 14195 NM_002170.2 GE55306 DISCOVERY 2.811950684 7.21875 14 2183 AA861218.1 GE80525 DISCOVERY 3.408691406 8.622650146 15 22675 BX117842.1 GE516913 DISCOVERY 81.83721924 204.4117737 16 3858 AI283196.1 GE59314 DISCOVERY 9.184204102 22.61538696 17 8431 NM_021081.3 GE56485 DISCOVERY 7.641021729 18.609375 18 18829 NM_002958.1 GE81500 DISCOVERY 6.883331299 15.3157959 19 8440 NM_016232.4 GE79419 DISCOVERY 7.225799561 15.71429443 20 3642 AW594132.1 GE543964 DISCOVERY 1627.052002 3306.942871 21 38566 NM_031273.1 GE84875 DISCOVERY 7.339630127 14.54998779 22 24910 NM_018556.2 GE60436 DISCOVERY 29.01333618 55.05334473 23 6329 BX116538.1 GE58014 DISCOVERY 37.80822754 67.53659058 2. Immune System Gene 1 25767 INCYTE GE59636 DISCOVERY 130.6451721 4927.383301 UNIQUE 2 29703 BX103139.1 GE59980 DISCOVERY 0.660003662 19.8302002 3 28615 AA504638.1 GE520093 DISCOVERY 2.739135742 20.69332886 4 30707 AI809890.1 GE54001 DISCOVERY 2.853668213 21.05661011 5 21607 NM_002960.1 GE80951 DISCOVERY 3.446289063 24.92727661 6 51904 H08511.1 GE893705 DISCOVERY 2.304870605 11.3684082 7 22254 NM_176891.2 GE80659 DISCOVERY 26.98571777 122.6947327 8 45170 BM671892.1 GE53155 DISCOVERY 38.31506348 85.25881958 9 22100 BX457477.2 GE616415 DISCOVERY 328.440918 608.4909058 Enhancement Ratio Description 1. Oncogene  1 45.89976479 zn87b11y5 Stratagene lung carcinoma 937218 cDNA clone IMAGE: 565149 5′ similar to contains Alu repetitive element; contains element MER22 repetitive element;  2 28.43685282 disrupted in renal carcinoma 1 (DIRC1)  3 26.91548242 Homo sapiens NEUROBLASTOMA COT 25- NORMALIZED cDNA clone CS0DC018YE24 3-PRIME  4 21.14346521 clone N11 NTera2D1 teratocarcinoma mRNA  5 12.79322267 NEUROBLASTOMA COT 25-NORMALIZED cDNA clone CS0DC011YD03 5-PRIME  6 11.8345813 HELA CELLS COT 25-NORMALIZED cDNA clone CS0DK007YM05 3-PRIME  7 9.469881078 zb91f05s1 Soares_parathyroid_tumor_NbHPA cDNA clone IMAGE: 320193 3′  8 9.348679048 breast carcinoma amplified sequence 1 (BCAS1)  9 9.115709518 yu38e06r1 Soares ovary tumor NbHOT cDNA clone IMAGE: 236098 5′ 10 6.360442594 RAS-like, family 11, member A (RASL11A) 11 5.87098009 RAB43, member RAS oncogene family (RAB43), mRNA 12 3.477979124 T-cell leukemia/lymphoma 6 (TCL6), transcript variant TCL6a1 13 2.567168067 B-cell translocation gene 4 (BTG4) 14 2.529607148 neuro-oncological ventral antigen 1 (NOVA1), transcript variant 1 15 2.497784939 endogenous retroviral family W, env(C7), member 1 (syncytin) (ERVWE1), mRNA 16 2.46242208 preferentially expressed antigen in melanoma (PRAME), transcript variant 3 17 2.435456365 Ras-associated protein Rap1 (RBJ) 18 2.225055752 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) (MYCN) 19 2.174748179 mab-21-like 2 (C elegans) (MAB21L2) 20 2.032475217 RAB2, member RAS oncogene family (RAB2) 21 1.982387061 RAS protein activator like 2, mRNA (cDNA clone IMAGE: 5399841), with apparent retained intron 22 1.897518589 platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog) (PDGFB), transcript variant 2 23 1.786293486 mutated in colorectal cancers (MCC) 2. Immune System Gene 1 37.71577029 tumor necrosis factor (TNF superfamily, member 2) (TNF) 2 30.04559116 chemokine (C—X—C motif) ligand 9 (CXCL9) 3 7.554692722 IL3-UT0117-080301-496-D07 UT0117 Homo sapiens cDNA 4 7.378787069 immunoglobulin superfamily, member 4C (IGSF4C) 5 7.233077713 colony stimulating factor 1 (macrophage) (CSF1), transcript variant 1 6 4.932341181 interleukin 1 family, member 8 (eta) (IL1F8), transcript variant 1 7 4.546654408 interleukin 4 (IL4), transcript variant 1 8 2.225203663 transforming growth factor beta 1 induced transcript 1 (TGFB1I1) 9 1.852664734 IL5-CI0001-181000-178-f03 CI0001 Homo sapiens cDNA B. Suppressed Gene Expression Expression Amount Amount before after Treatment Treatment with with Gene Gene the the No. Gene Name Bank Peptide Peptide 1. Cancer Sup- pressing Gene 1 35687 BX108016.1 GE82100 DISCOVERY 103.9224243 0.225219727 2 36334 BF433017.1 GE82101 DISCOVERY 341.440918 2.079650879 3 37600 AW979088.1 GE62125 DISCOVERY 982.9432373 8.42855835 4 30909 NM_018412.2 GE79080 DISCOVERY 987.7108154 11.47369385 5 8280 BC063301.1 GE62103 DISCOVERY 758.4154053 9 6 11438 NM_152271.2 GE82058 DISCOVERY 1207.091431 14.74417114 7 11261 NM_018559.2 GE56225 DISCOVERY 379.1338501 6.257141113 8 50395 NM_004480.3 GE79552 DISCOVERY 470.2086792 8.763153076 9 52111 AK056875.1 GE61359 DISCOVERY 593.6870728 13.6000061 2. Immune System Gene 1 20881 NM_001482.1 GE61110 DISCOVERY 6599.050293 38.16882324 2 47973 NM_004394.1 GE81458 DISCOVERY 1480.68396 9.638885498 3 10791 W00901.1 GE80378 DISCOVERY 517.4453125 3.42855835 4 15907 BU899259.1 GE80960 DISCOVERY 912.8641357 6.363647461 5 16756 AI707455.1 GE81522 DISCOVERY 395.0359497 2.80645752 6 34736 AV736303.1 GE80100 DISCOVERY 3974.221191 29.0786438 7 39991 AI252940.1 GE61199 DISCOVERY 2414.748779 17.77420044 8 41726 AI453596.1 GE58507 DISCOVERY 4512.05127 33.50683594 9 45767 BG190549.1 GE79374 DISCOVERY 2359.263184 18.125 10 42614 AW296107.1 GE81744 DISCOVERY 626.2438965 4.848480225 11 20551 AK024566.1 GE86210 DISCOVERY 2811.726807 22.93103027 12 38103 NM_173695.1 GE61298 DISCOVERY 703.8875122 5.777770996 13 37068 AW971488.1 GE58019 DISCOVERY 6960.161133 57.38461304 14 4899 BX115183.1 GE80961 DISCOVERY 536.4679565 4.5 15 43032 BC013284.2 GE58028 DISCOVERY 519.3710938 4.445770264 16 42813 NM_001763.1 GE58393 DISCOVERY 4447.713379 38.51351929 17 22326 NM_014396.2 GE82907 DISCOVERY 1102.541626 9.742858887 18 37445 NM_007068.2 GE60016 DISCOVERY 1671.436768 14.875 19 22890 BX119833.1 GE86305 DISCOVERY 1112.63855 9.902160645 20 16478 NM_020530.3 GE54528 DISCOVERY 663.3167725 6.018188477 21 52539 AI133415.1 GE82473 DISCOVERY 17346.24609 158.6896667 22 41803 NM_178313.1 GE735470 DISCOVERY 89.23596191 0.820007324 23 41817 NM_016292.1 GE79992 DISCOVERY 10618.77539 98.42697144 24 18884 NM_015604.2 GE57715 DISCOVERY 339.244873 3.387084961 25 8922 NM_024077.2 GE57504 DISCOVERY 10964.4248 110.8526306 26 24969 AV752332.1 GE60370 DISCOVERY 2446.138672 25.71212769 27 8634 BC043004.2 GE566269 DISCOVERY 947.6875 10.19564819 28 38879 NM_182832.1 GE476899 DISCOVERY 2933.067871 31.93505859 29 49586 BG289120.1 GE58687 DISCOVERY 248.4822998 2.715911865 30 11651 AW850450.1 GE85351 DISCOVERY 1144.841431 12.83999634 31 53172 NM_022749.4 GE59644 DISCOVERY 577.4226074 6.560333252 32 49622 CB052158.1 GE59962 DISCOVERY 3421.524414 39.68115234 33 22868 NM_152285.1 GE79323 DISCOVERY 539.8291016 6.525421143 34 23827 BG221408.1 GE59882 DISCOVERY 2403.98877 29.7802124 35 18139 AA938869.1 GE890404 DISCOVERY 2660.284912 33.11999512 36 22789 BX104097.1 GE61439 DISCOVERY 731.80896 9.725494385 37 11236 NM_003937.1 GE58794 DISCOVERY 4738.49707 65.4675293 38 10204 AW197778.1 GE81039 DISCOVERY 696 9.714294434 39 49170 AA577911.1 GE61247 DISCOVERY 713.4000244 10.09091187 40 42474 AW137161.1 GE62833 DISCOVERY 1164.558472 17.03775024 41 52342 NM_006147.2 GE54992 DISCOVERY 1200.007446 18.71737671 42 42573 NM_022648.2 GE79383 DISCOVERY 953.4078369 15.75 43 24440 NM_182936.1 GE592149 DISCOVERY 14 0.234771729 45 24440 NM_182936.1 GE592149 DISCOVERY 14 0.234771729 46 24440 NM_182936.1 GE592149 DISCOVERY 14 0.234771729 47 46186 NM_032746.1 GE82057 DISCOVERY 1407.181763 25.55319214 48 39926 INCYTE GE82589 DISCOVERY 17409.49609 317.7109375 UNIQUE 49 47689 AK097380.1 GE61992 DISCOVERY 3803.877441 72.08435059 50 12812 CF135919.1 GE55506 DISCOVERY 1176.72522 24.53225708 51 15994 R25284.1 GE79364 DISCOVERY 11397.33301 239.4736938 52 52071 BM698907.1 GE86827 DISCOVERY 415.7030029 9.42855835 53 40727 BG573885.1 GE55241 DISCOVERY 1204.090942 28.16665649 54 11635 AI376607.1 GE80662 DISCOVERY 106.1363525 2.5 55 4403 N26032.1 GE56202 DISCOVERY 891.694458 21.96551514 56 14949 BX110547.1 GE81738 DISCOVERY 1020.042969 25.26760864 57 51004 BM670853.1 GE59115 DISCOVERY 345.9705811 8.725006104 58 17177 AI274757.1 GE54607 DISCOVERY 1033.54187 26.16049194 59 9938 NM_152288.1 GE87537 DISCOVERY 11.78378296 0.299987793 60 21487 NM_006861.4 GE58813 DISCOVERY 387.6266479 9.909088135 61 37834 BU608350.1 GE57222 DISCOVERY 545.7602539 14.10638428 62 42661 C04533.1 GE60353 DISCOVERY 1506.339355 39.14474487 63 42680 AW449970.1 GE80314 DISCOVERY 1616.487305 42.02856445 Suppression Ratio Description 1. Cancer Sup- pressing Gene 1 461.4268293 heat shock protein 75 (TRAP1) 2 164.1818449 heat shock 70 kDa protein 14 (HSPA14) 3 116.6205651 AHA1, activator of heat shock 90 kDa protein ATPase homolog 1 (yeast) (AHSA1) 4 86.08481528 tumor rejection antigen (gp96) 1 (TRA1) 5 84.26837836 brain specific protein (CGI-38) 6 81.86905991 serologically defined breast cancer antigen 84 (SDBCAG84), transcript variant 1 7 60.59218471 breast cancer metastasis suppressor 1 (BRMS1) 8 53.65747638 Ras suppressor protein 1 (RSU1), transcript variant 1 9 43.65344164 leukemia cell normalized cDNA library cDNA clone LEU1757_26_C2 2. Immune System Gene 1 172.891112 natural killer cell transcript 4 (NK4) 2 153.615681 CD8 antigen, beta polypeptide 1 (p37) (CD8B1), transcript variant 5 3 150.9221252 HLA-B associated transcript 5 (BAT5) 4 143.4498283 intercellular adhesion molecule 2 (ICAM2) 5 140.7596399 major histocompatibility complex, class I, E (HLA-E) 6 136.6714768 adhesion molecule AMICA (AMICA) 7 135.8569567 lymphotoxin beta (TNF superfamily, member 3) (LTB), transcript variant 2 8 134.660619 apoptosis-associated speck-like protein containing a CARD (ASC), transcript variant 1 9 130.1662446 HLA-B associated transcript 1 (BAT1), transcript variant 1 10 129.1629268 killer cell lectin-like receptor subfamily K, member 1 (KLRK1) 11 122.6166802 IL2-inducible T-cell kinase (ITK) 12 121.8268278 tumor necrosis factor receptor superfamily, member 7 (TNFRSF7) 13 121.2896762 immediate early response 2 (IER2) 14 119.2151015 interferon (alpha, beta and omega) receptor 2 (IFNAR2), transcript variant 1 15 116.8236465 interferon, gamma-inducible protein 16 (IFI16) 16 115.4844704 granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) (GZMA) 17 113.1640763 immune associated nucleotide 2 (hIAN2) 18 112.365497 mal, T-cell differentiation protein (MAL), transcript variant d 19 112.3632094 NK inhibitory receptor precursor (NKIR) 20 110.2186771 tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), transcript variant 1, mRNA 21 109.3092351 major histocompatibility complex, class II, DR alpha (HLA-DRA) 22 108.8233718 programmed cell death 2 (PDCD2), transcript variant 2 23 107.8848128 major histocompatibility complex, class I, F (HLA-F) 24 100.1583595 (clone 38-1) MHC class I mRNA fragment. 25 98.90991981 interferon induced transmembrane protein 1 (9-27) (IFITM1) 26 95.13559911 CD69 antigen (p60, early T-cell activation antigen) (CD69) 27 92.9501962 perform 1 (pore forming protein) (PRF1) 28 91.84476247 interferon stimulated gene 20 kDa (ISG20) 29 91.49129726 interleukin-1 receptor-associated kinase 4 (IRAK4) 30 89.16213062 implantation-associated protein (DKFZp564K142) 31 88.01726761 transforming growth factor, beta 1 (Camurati-Engelmann disease) (TGFB1) 32 86.22542976 intercellular adhesion molecule 3 (ICAM3) 33 82.72708991 lymphocyte cytosolic protein 2 (SH2 domain containing leukocyte protein of 76 kDa) (LCP2) 34 80.72436613 major histocompatibility complex, class II, DM alpha (HLA-DMA) 35 80.32262392 major histocompatibility complex, class II, DQ alpha 1 (HLA-DQA1) 36 75.24645339 CASP8 and FADD-like apoptosis regulator (CFLAR) 37 72.37934777 natural killer cell group 7 sequence (NKG7), mRNA 38 71.64699451 interferon-related developmental regulator 1 (IFRD1) 39 70.69728028 interferon gamma receptor 2 (interferon gamma transducer 1) (IFNGR2) 40 68.35165764 programmed cell death 6 (PDCD6) 41 64.11194608 apoptosis-related protein PNAS-1 (FLJ39616) 42 60.53383092 T-cell activation protein (PGR1) 43 59.63239308 THYMUS cDNA clone CS0CAP007YL06 5- PRIME 45 59.63239308 THYMUS cDNA clone CS0CAP007YL06 5- PRIME 46 59.63239308 THYMUS cDNA clone CS0CAP007YL06 5- PRIME 47 55.06872703 cell death-regulatory protein GRIM19 (GRIM19) 48 54.79665331 thymosin-like 6 (TMSL6) 49 52.7698094 killer cell lectin-like receptor subfamily B, member 1 (KLRB1) 50 47.96644744 ovarian carcinoma immunoreactive antigen (OCIA) 51 47.5932568 beta-2-microglobulin (B2M) 52 44.08977359 interferon (alpha, beta and omega) receptor 1 (IFNAR1) 53 42.74880629 linker for activation of T cells (LAT) 54 42.45454102 thymosin, beta 4, Y-linked (TMSB4Y) 55 40.595199 programmed cell death 4 (neoplastic transformation inhibitor) (PDCD4), transcript variant 2 56 40.36958872 programmed cell death 10 (PDCD10), transcript variant 1 57 39.65276092 tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10) 58 39.50773832 tumor necrosis factor superfamily, member 5-induced protein 1 (TNFSF5IP1) 59 39.28087487 interleukin 17D (IL17D) 60 39.11829653 programmed cell death 2 (PDCD2), transcript variant 1 61 38.68888322 proteasome (prosome, macropain) 26S subunit, non-ATPase, 1 (PSMD1) 62 38.4812664 proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional protease 2) (PSMB9), transcript variant 1 63 38.46163498 proteasome (prosome, macropain) 26S subunit, ATPase, 4 (PSMC4), transcript variant 1

This experiment revealed the facts described below, and it was clarified that the artificially synthesized peptide also significantly influenced the expression of human cancer-related gene to induce cancer, as in the peptide derived from the original cell line.

A. Enhanced Gene

1. Oncogene

Expression of oncogenes of cranial neuroblastoma, lung cancer, parotid gland tumor, breast cancer, colon cancer, renal cancer, ovarian cancer, melanoma, T-cell leukemia, B-cell leukemia, and so on were enhanced in the range of 47 to 1.7 times the baseline expression thereof. Expression of carcinogenic genes such as RAS, RAB, v-myc, and mab were also enhanced in the range of 6.7 to 1.7 times the baseline expression thereof. Additionally, expression of cell growth factor such as a platelet-derived growth factor was enhanced.

2. Immune System Gene

Expression of TNF, which is an inflammatory factor, was not enhanced, but expression of most of important factors as an operating factor for the humoral immunity mechanism, which is the opposite side of anti-tumor immunity, was enhanced.

B. Suppressed Gene

1. Cancer-Suppressing Gene

Every cancer-suppressing gene was significantly suppressed: Expression of heat shock protein relating to cancer antigen-induction was suppressed in the range of 1/461 to 1/116 of the baseline expression thereof, and expression of a tumor rejection antigen was suppressed in the range of 1/86 to 1/81 of the baseline expression thereof. Expression of a gene relating to inhibition of metastasis of breast cancer was suppressed to 1/60, expression of a gene relating to inhibition of leukemia was suppressed to 1/43, and expression of RAS suppressor protein gene was suppressed to 1/43.

2. Immune System Gene

The expression of genes relating to anti-tumor immunity was highly suppressed as follows:

suppression of a gene relating to construction of thymus tissues or production of a thymic hormone;

suppression of a function and a construction factor of T-cell which is important in inhibition of cancer;

suppression of a function and construction of NK-cell;

suppression of production of a factor for killer-cell-secretion;

suppression of an apoptosis factor relating to cellular suicide;

suppression of a factor relating to cell adhesion in cell apoptosis;

suppression of production of antineoplastic interferon; and

suppression of expression of a cell organelle, an enzyme system, and a major histocompatibility complex (MHC) relating to induction of a cancer antigen.

(b) The peptide obtained in Example 3 was added to a culture medium (10 to 100 μg peptide/1×10⁷ monocytes) for human normal peripheral blood monocytes extracted from subjects shown in Table 5 below, and they were cocultured for 7 hr. Then, total mRNA was extracted from the monocytes, and the amount of the total mRNA was measured and compared to that of the total mRNA before the treatment with the peptide. Table 5 shows the results.

TABLE 5 Effect of the artificially synthesized peptide on total mRNA amount of peripheral blood monocytes pre post total total RNA RNA post/ reduction NAME (μg/ml) (μg/ml) pre (%) ratio (%) H.S. 45.875 0.958 2 98 Healthy subject (smoker) M.S. 20.779 9.188 44.2 55.8 Healthy subject (smoker) E.E. 69.622 7.735 11.1 88.9 Healthy subject (side-stream smoke) S.G. 61.145 16.562 27 73 Healthy subject H.T. 66.804 14.742 22 78 Healthy subject (side-stream smoke) M.O. 58.076 19.551 33.6 66.4 Healthy subject (side-stream smoke) M.A. 52.318 17.681 33.7 66.3 Healthy subject K.K. 8.078 1.757 21.7 78.3 Cancer patient received chemotherapy and radiotherapy Samples are equivalent to 1 × 10⁷ peripheral blood monocytes. Treatment time with the peptide: 7 hr The processes for collecting total RNA are the same as those in Example 4.

With referred to the results shown in Table 5, the total mRNA in H. S. and E. E. were significantly decreased. H. S. was an addicted smoker who has smoked for more than ten years. With respect to M. S., the reduction ratio of total mRNA was low, but the pre-total mRNA amount itself was also low, which has been caused by smoking over a long period of time. E. E. was not a smoker, but father of E. E. was an addicted smoker. Therefore, it was assumed that the decrease in the total mRNA was caused by passive smoking over a long period of time. Both H. T. and M. O. were passive smokers, and the reactivity to the peptide was accelerated in proportion to degree of the passive smoking (H. T.>M. O.). In K. K., the reduction ratio was low and the pre-total mRNA amount was significantly small. This subject was a cancer patient who has already received an anticancer agent and irradiation at the maximum possible dose. Therefore, the general gene translation was significantly impaired; which caused the result entirely different from that in healthy subjects. The results shown in Table 5 show that the cancer diagnosis can be efficiently performed by analyzing the changing ratio of total mRNA amounts before and after the treatment with the peptide of the present invention.

Example 5 Detection of the Peptide of the Present Invention in Cancer Cells and Influence of the Peptide on Normal Cells

In order to examine influences at a gene level of the peptide on normal cells, RT-PCR was performed by adding the peptide artificially synthesized in Example 3 to the normal cells.

(Method and Result)

(1) Normal cells were treated with the artificially synthesized peptide for 7 hr as follows:

No. of Cells Peptide Amount Kidney mesangium cell: 1.25 × 10⁶ 12.5 μg (ACBRI-1376; Applied Cell Biology Research Institute) Skin fibroblast cell:  4.0 × 10⁶ 40.0 μg (Catlog2F0-C25; Cell Systems) Pancreatic epithelial cells: 1.05 × 10⁷ 105.0 μg  (ACBRI515; Applied Cell Biology Research Institute) Peripheral blood monocytes: H.S. 1.25 × 10⁷ 12.5 μg M.S. 1.20 × 10⁷ 12.0 μg E.E. 1.40 × 10⁷ 14.0 μg (Kidney mesangium cells, skin fibroblast cells, and pancreatic epithelial cells: 100 μg peptide/1.0 × 10⁷ cells, peripheral blood monocytes: 10 μg peptide/1.0 × 10⁷ cells)

Then, total RNA was extracted from the above-treated normal cells, untreated normal cells, and squamous-cell carcinoma cells by using an RNA extraction kit (QIAGEN). The numbers of the treated cells and the untreated cells were the same.

No. of Cells RNA Amount Kidney mesangium cell: 1.25 × 10⁶ 30 μg (ACBRI-1376; Applied Cell Biology Research Institute) Skin fibroblast cell:  4.0 × 10⁶ 30 μg (Catlog2F0-C25; Cell Systems) Pancreatic epithelial cells: 1.05 × 10⁷ 30 μg (ACBRI515; Applied Cell Biology Research Institute) Peripheral blood monocytes: H.S. 1.25 × 10⁷ 60 μg M.S. 1.20 × 10⁷ 60 μg E.E. 1.40 × 10⁷ 60 μg Cervical carcinoma cells (HeLa): 1.60 × 10⁷ 60 μg

Reverse transcription was performed by using 30 μg of each extracted total RNA to generate cDNA pools.

PCR was conducted using each of the cDNA pools as templates.

The condition for the PCR was as follows:

PCR buffer: 6 μl

dNTP: 2 μl

Primer Fw (11 amino acids or 7 amino acids): 1 μl

Primer Rv (oligo dT): 1 μl

dH2O: 9.5 μl

taq: 0.5 μl

Sample (RT products): 10 μl

(Total: 301)

PCR: 45 cycles

Initial denaturation: at 94° C. for 5 min

Denaturation: at 94° C. for 30 sec

Annealing: at 55° C. for 30 sec

Extension: at 72° C. for 1 min

(2) Each of the PCR products was applied to electrophoresis and detected by using a fluorescence label.

(a) FIGS. 2A and 2B show the results when the full-length peptide (11 amino acids) of the present invention was used as the primer in the PCR.

The Results are as Follows:

(i) Lanes 2 and 3 in FIG. 2A show the results of original UTC-8 cell samples which were not treated with the peptide of the present invention. The original UTC-8 cell samples stably expressed the gene of the preset invention.

Lane 4 in FIG. 2A shows the result of a cervical carcinoma cell sample, wherein a smear was observed. This means that the sample includes various sizes of genes which are complementary to the gene encoding the peptide of the present invention. Namely, the expression pattern of the gene was significantly changed in the cervical carcinoma cell sample by the treatment with the peptide of the present invention. Therefore, samples showing such a reaction pattern are thought to have a tendency of canceration.

(ii) On the basis of the results above, samples on each lane were examined. In some samples (Lanes 5 and 6 of FIG. 2A, lanes 4 and 5, lanes 6 and 7, and lanes 8 and 9 in FIG. 2B), the original stable gene pattern was changed to a pattern having a broadened smear by the treatment with the peptide of the present invention. Such samples were determined to have a tendency of canceration. Actually, the blood sample of the lanes 5 and 6 of FIG. 2A was obtained from a young man who had smoked for more than 10 years. Though the samples were normal cells, they had a high sensitivity to the peptide. It is thought that such a high sensitivity is caused by that these cells were, as in the peptide of the present invention, extracted from epithelial cells and were derived from a tissue in an early stage. Therefore, the gene expression is not fixedly stable, and the unstable gene expression is involved in the acuity sensitivity against a highly disturbing factor from the outside such as the peptide of the present invention. (iii) On the contrary, in the sample of lanes 2 and 3 of FIG. 2B, gene mutation was hardly induced even if the sample was treated with the peptide of the present invention. This sample is thought to have a characteristic to rarely get cancer. Actually, the sample was obtained from a healthy young man in twenties. (b) FIGS. 3A and 3B show the results when a DNA encoding an amino acid sequence (7 amino acids) at positions 1-7 of the peptide of the present invention was used as a primer in the PCR.

The Results are as Follows:

(i) Smears were observed in an UTC-8 cell sample shown in lane 3 of FIG. 3A and in a cervical carcinoma cell sample shown in lane 4 of FIG. 3A, as in the results when the full sequence was examined in the above-mentioned (a).

(ii) In the samples of lanes 2 and 3, lanes 4 and 5 of FIG. 3B, smears were not observed even if the samples were treated with the peptide of the present invention, and normal patterns were observed.

(iii) In the samples of lanes 5 and 6, lanes 7 and 8 of FIG. 3A and lanes 6 and 7, lanes 8 and 9 of FIG. 3B, gene expression patterns were significantly changed before and after the treatment with the peptide of the present invention.

As shown above, it was clarified that a tendency of canceration in normal cells can be determined by comparing changes in gene patterns induced by treating the cells with the peptide of the present invention.

In particular, when the PCR was performed by using the 11-amino acid primer (full sequence), a difference in the band patterns was observed in the peripheral blood sample (H. S.) and the skin fibroblast sample, and a difference in a position having a high concentration was observed in the kidney mesangium sample and the pancreatic epithelial sample, though it was a smear. When the PCR was performed by using the 7-amino acid primer (partial sequence), the band pattern of the pancreatic epithelial sample treated with the peptide for 7 hr was similar to that of the UTC-8 cell sample used as a control, and a difference in the band patterns was observed in the pancreatic epithelial sample by the treatment with the peptide for 7 hr.

A possible explanation for causes of such significant effects is that the UTC-8 cell is a cancer cell of epithelium and the peptide of the present invention is derived from UTC-8 cell line.

Therefore, epithelial cells are the most suitable samples for the measurement of a tendency of canceration using the peptide of the present invention, but it was proved that non-epithelial cell samples including peripheral blood, which can be most easily obtained in clinical practice, were useful for the diagnosis of a gene change in the full sequence.

Example 6 Exhaustive Gene Analysis of 55000 Genes by Using a DNA Chip System (Codelink System)

Characteristics of the peptide of the present invention were clarified by conducting the analysis described below in order to examine effects of the peptide on gene expression of normal cells.

(1) Preparation of target cRNA

Normal lymphocytes were treated with the peptide of the present invention as in Examples 4 and 5, and the gene expression was compared to that of untreated lymphocytes by using a DNA chip system (Codelink System). In this examination, cRNA derived from bacteria was used as a positive control. Total RNA was extracted by using the QIAGEN RNA extraction kit used in Example 5. The total RNA (5 to 10 μg) was subjected to reverse transcription using an oligo-dT primer having a T7 RNA polymerase promoter region as a reverse primer to generate a pool of cDNAs complementary to the mRNAs. The condition for the reverse transcription was the same as that in Example 5. After second-strand cDNA synthesis, the treatment with T7 RNA polymerase and biotinylated rNTPs was performed in vitro transcription (IVT) reaction at 37° C. for 14 hr to obtain biotin-labeled cRNA. The amount of the cRNA was measured with a spectrophotometer to confirm at least 10 μg of the cRNA.

(2) Hybridization to Codelink slide

The cRNA (10 μg) obtained in the above (1) was fragmentized and mixed with hybridization buffer, and then thermally denatured. This mixture solution was poured into a Codelink slide on which 55000 genes were immobilized, and a hybridization reaction was performed at 37° C. for 18 hr using an INNOVA 4080 shaker.

(3) Staining and detection

The hybridization chamber was removed and the plate was washed at 46° C. for 1 hr. The hybridization was detected with Cy5-streptavidin (1:500 dilution, at room temperature for 30 min). The plate was further washed at a room temperature for 5 min 5 to 6 times and then washed with 0.05% Tween 20. The plate was dried by using a centrifuge for titer plates.

(4) The plate was scanned with an Arra WORK scanner with a PMT value of 600 at a resolution of 10 μm.

(5) The result was analyzed by exclusive software for the Codelink analysis. The software used was 1. Batch Submission 2 and 2. Codelink Expression 2.

Cancer-inducing expression status of cancer-related genes was observed in this experiment too, as in the results shown in Tables 1, 2, and 3.

Example 7 Examination of Antibody Against the Peptide for Cytotoxic Activity on Human Cancer Cells

Monoclonal antibody (prepared by using BALB/c mouse) against the peptide of the present invention was examined for cytotoxic activity on various human cancer cell lines shown in FIG. 4: Each cancer cell line was seeded in a 96-well plate at 1×10⁴ cells/well, and cultured in 5% CO₂ for 4 days to confirm that the cancer cells proliferated and adhered to the bottom surface of the plate so as to cover more than 80% of the surface area. The culture medium was removed by aspiration, and then 30 μl of the monoclonal antibody was added to each well under ice-cooling so that the surfaces of the cancer cells were brought into contact with the culture medium containing the monoclonal antibody. Then, the cancer cells were cultured under ice-cooling for 1 hr. As a control, each cancer cell line was also cultured in PBS buffer not containing the monoclonal antibody. Cytotoxic activity of the monoclonal antibody was examined by comparing the cancer cells treated with the monoclonal antibody with those of the control, and viability of the cell was determined by Trypan Blue staining. In the control, no substantial detachment of the cells from the bottom surface of the plate was observed; hence, cytotoxicity was not observed. On the contrary, in the cancer cells treated with the monoclonal antibody against the peptide of the present invention, detachment of the cells was observed in cancer cells shown in FIG. 4; hence, cytotoxic activity of the monoclonal antibody was observed. In addition, the detachment of the cancer cells was immediately induced by the addition of the monoclonal antibody, and the cancer cells were killed by the activity of the monoclonal antibody within only one hour. The photograph in the right of FIG. 4 shows human pancreatic cancer cells stained with FITC-labeled monoclonal antibody No. 1 of the present invention. These human pancreatic cancer cells died after this treatment.

Example 8 Immunosuppressive Agent Reducing Rejection in Transplantation

The experiment using the DNA chip proved that the peptide (SEQ ID NO: 1) had an ability to activate a cancer-related gene, and further suggested clinical usefulness of the cellular immunity-inhibition activity of the peptide. Then, the cellular immunity-inhibition activity of the peptide was examined from the viewpoint of activity for preventing rejection in skin transplantation.

The peptide of the present invention was administered to mice who have received skin transplantation according to the schedule shown in Table 6.

TABLE 6 Days from Administration Transplantation Transplantation of Peptide −3 Administration −2 Administration −1 Administration 0 Operation 1 2 Administration 3 Administration 4 5 6 7 Administration 8 9 10 11 12 13 14 Administration 15 16 Subject: skin of the back of a C57Black/6N mouse was transplanted to the back of a BALB/c mouse Administration route: subcutaneously administered into the neck Administration amount: 50 μg peptide/0.1 ml/mouse Number of the subject: 6

The results show that, in the cross-transplantation of skin of the back of a C57Black/6N mouse to the back of a BALB/c mouse, the rejection in the skin transplantation was suppressed and skin graft survival was prolonged by intermittently administering the peptide at a very low amount of 50 μg peptide/0.1 ml/mouse through a subcutaneous route which is a most gentle administration (see the progress shown by the graph in FIG. 5). Namely, in the control, skin rejection occurred 7 days after the skin transplantation and the transplanted skin was rejected and was lost within 10 days after the transplantation in all 6 mice as shown in a photograph (control G1) of mice at bottom right in FIG. 5. On the contrary, in the mice administered with the peptide, skin rejection occurred 12 days after the transplantation; which was a 5 day delay. Furthermore, the transplanted skin of one mouse was survived for 14 days after the transplantation as shown in a photograph (treatment G1) at upper right in FIG. 5. Though the transplanted skin was lost in all mice on the 15th day from the date of the transplantation, a delay in the rejection in skin transplantation was observed.

Therefore, it was proved that the peptide can be used as an immunosuppressive agent by investigating administration dosage and route to increase efficiency thereof. 

1. A polynucleotide consisting of a nucleotide sequence encoding the eleven contiguous amino acids of the amino acid sequence of a peptide having an ability to activate a cancer-related gene, wherein the peptide is obtained from cell membrane surfaces of human squamous-cell carcinoma cells and consists of the amino acid sequence represented by SEQ ID NO:
 1. 